Using the formation of a virus particle (virion) from SV40 minichromosomes as a model for the generation of repressive chromatin by nucleosome remodeling, we will characterize how the combination of nucleosome location and associated histone modifications controls gene expression. Our central hypothesis is that the nucleosome location and the histone modifications associated with the nucleosome directly contribute to reorganization. The specific aims are (1) To characterize the changes in nucleosome positioning and corresponding histone modifications during the encapsidation of minichromosomes late in infection and (2) To determine which transcriptional regulatory sequences direct nucleosome positioning and histone modifications during encapsidation. For comparison purposes bulk nucleosome locations will be determined from micrococal nuclease digestion of chromatin from minichromosomes and disrupted virions. The location of nucleosomes containing specific histone modifications along with all of the other nucleosome positions in each sample of chromatin will be obtained using our modified ChIP-Seq procedure, which we developed. Intact SV40 chromatin will be immune selected with antibody to a particular histone modification bound to agarose, the ChIP chromatin purified on the agarose, and fragmented by micrococcal nuclease digestion. The DNA present in the agarose-bound chromatin fragments and the free fragments will be separately purified, libraries prepared, and paired-end sequenced using an Illumina MiSeq. The location of nucleosomes will then be determined from a bioinformatics analysis of the sequencing reads in the parallel libraries. SV40 chromatin will be obtained from wild-type and mutant minichromosomes and encapsidation intermediates and recombinant constructs containing specific regulatory elements within a defined chromatin reporter region. The changes in nucleosome location and histone modifications will then be correlated with the process of encapsidation and the presence of specific DNA elements. The proposed studies are innovative because for the first time we will be characterizing both nucleosome location and histone modifications in the same nucleosomes while remodeling to repress transcription is occurring as part of virion formation. The work is also significant since linking histone modifications to nucleosome location would advance our understanding of how nucleosome location is regulated. Because SV40 is also a model for closely related human pathogens understanding epigenetic repression by nucleosome positioning may lead to novel therapeutic approaches to address the disease caused by the pathogens.